| Bacteria in their normal environment are constantly confronted with stresses such as temperature, osmolarity, and pH changes. In order to survive these conditions, bacteria have evolved adaptive mechanisms. The work of this dissertation focuses on characterizing aspects of the stress responses of two Gram positive bacteria: Streptococcus pyogenes and Bacillus subtilis . Specifically, I examined the activation of the principal Heat Shock chaperones (DnaK, GroESL) in S. pyogenes and physical stress induction of the sigmaB regulon in B. subtilis.; Among the stress inducible systems characterized in bacteria, the heat induced expression of the DnaK and GroEL molecular chaperones is one of the most intensely studied. S. pyogenes encodes dnaK and groEL as well as HrcA, a negative regulator of dnaK and groESL expression in other gram-positive bacteria.; To investigate whether HrcA is also a regulator of dnaK and groEL in S. pyogenes, a strain of this bacterium was created lacking HrcA. The RNA levels of both dnaK and groEL were elevated in the HrcA mutant strain even in the absence of stress. An additional increase in expression was demonstrated in the HrcA- strain upon exposure to heat stress. This indicates that HrcA is a negative regulator of dnaK and groEL expression in S. pyogenes, but that there is an additional level of regulation controlling the synthesis or stability of the RNA messages of these operons in stressed cells.; The general stress regulon of Bacillus subtilis is controlled by the transcription factor sigmaB. Diverse physical stresses activate sigmaB through a phosphatase/kinase cascade that ultimately causes sigmaB's release from a protein inhibitor. RsbT, a kinase and phosphatase activator is the key positive regulator of the sigmaB pathway. In unstressed cells, RsbT is complexed to an inhibitory protein (RsbS). Following exposure to stress, RsbT inactivates RsbS by phosphorylation and then binds and activates an additional regulator (RsbU) which drives the sigmaB release.; Using alanine-scanning mutagenesis 17 mutations were created throughout the length of RsbT at sites where charged amino acids are clustered. In addition, an 18th mutation was created by deleting the 14 amino acid carboxy terminus of the protein. Using Western blot analyses, we determined that 5 of the rsbT mutations altered sites that were essential for the protein product to accumulate in B. subtilis. Activity assays identified 7 of the remaining mutations as changes that did not alter RsbT's known activities, and 6 others that defined sites essential for RsbT's ability to respond to stress.; The 6 RsbTs that failed to activate sigmaB were studied in more detail. None of these RsbTs were able to activate RsbU in an assay where the activation was independent of the normal stress induction signals. 3 of the mutant RsbTs could, however, compete for the RsbS inhibitor and allow a population of wildtype RsbT proteins to become active.; When the mutant RsbT proteins were examined for potential changes in their binding properties in the yeast dihybrid system, the inactive RsbTs displayed a consistent failure to interact with RsbS, a property that was expected to make them constitutively active rather than inactive. The data suggest that RsbT's ability to interact with RsbS may be coincident with its ability to activate RsbU, perhaps due to a common region in RsbT that is needed for both interactions. |
